CN115166067A - Peptide fragment composition for relatively quantitatively analyzing 10-formyltetrahydrofolate dehydrogenase (ALDH) 1L1 of pig and application thereof - Google Patents

Peptide fragment composition for relatively quantitatively analyzing 10-formyltetrahydrofolate dehydrogenase (ALDH) 1L1 of pig and application thereof Download PDF

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CN115166067A
CN115166067A CN202210587648.8A CN202210587648A CN115166067A CN 115166067 A CN115166067 A CN 115166067A CN 202210587648 A CN202210587648 A CN 202210587648A CN 115166067 A CN115166067 A CN 115166067A
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peptide fragment
aldh1l1
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CN115166067B (en
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高杰
张名媛
顾先红
朴香淑
郝月
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Institute of Animal Science of CAAS
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Abstract

The application provides a peptide fragment composition, a method and a kit for relatively quantitatively analyzing 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 of a pig. Relatively quantitatively analyzing a peptide fragment composition of the pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1, wherein the peptide fragment composition comprises a first peptide fragment, a second peptide fragment and a third peptide fragment; the amino acid sequence of the first peptide segment is shown as SEQ ID NO. 1; the amino acid sequence of the second peptide segment is shown as SEQ ID NO.2 and the amino acid sequence of the third peptide segment is shown as SEQ ID NO. 3. The porcine ALDH1L1 can be quickly and efficiently quantitatively detected on the protein level, and the detection flux and efficiency of the porcine ALDH1L1 are improved. Compared with the Western Blot method based on the antibody, the method has stronger specificity.

Description

Peptide fragment composition for relatively quantitative analysis of porcine 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 and application thereof
Technical Field
The application relates to the technical field of food detection, in particular to a peptide fragment composition for relatively quantitatively analyzing 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 of a pig and application thereof.
Background
In the swine industry, oxidative stress easily causes economic losses. Economic losses due to oxidative stress are mainly manifested by damage to the pig liver, which in turn affects the health of the pig and reduces the quality of the pig product (e.g. pork, etc.). Oxidative stress is often caused by stimulation by stressors, disrupting the oxidation-antioxidant balance system in the pig, and causing a dramatic increase in oxidative free radicals and their Reactive derivatives (ROS), among others.
ALDH1L1 is a member of the aldehyde dehydrogenase superfamily, a regulator of folate metabolism. ALDH1L1 is also known as 10-Formyltetrahydrofolate Dehydrogenase (FDH). Mainly used for catalyzing the conversion of 10-formyl tetrahydrofolic acid into tetrahydrofolic acid and CO 2 Is by NADP + Is required for the reaction-dependent catalysis of DNA nucleotide biosynthesis of Tetrahydrofolate (THF) synthesis from 10-formylTHF. Has regulation and control function and higher research value in the process of oxidation stress related physiology and pathology.
Therefore, the research on the relative quantitative analysis method of the pig 10-formyltetrahydrofolate dehydrogenase ALDH1L1 has important significance.
Disclosure of Invention
In view of the above, the present application aims to provide a peptide fragment composition for relatively quantitatively analyzing porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1 and an application thereof.
In view of the above, the present application provides a peptide fragment composition for relatively quantitative analysis of porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1, comprising a first peptide fragment, a second peptide fragment and a third peptide fragment;
wherein the amino acid sequence of the first peptide segment is shown as SEQ ID NO. 1; the amino acid sequence of the second peptide segment is shown as SEQ ID NO. 2; the amino acid sequence of the third peptide segment is shown as SEQ ID NO. 3.
In some of these embodiments, the parent ion of the first peptide fragment is 770.89m/z, the daughter ions are 1054.57m/z,907.50m/z and 737.39m/z, and the corresponding collision energy of the daughter ions is 27V; the parent ions of the second peptide fragment are 682.35m/z, the daughter ions are 1135.57m/z,949.51m/z and 878.47m/z, and the corresponding collision energy of the daughter ions is 27V; the parent ion of the third peptide segment is 812.89m/z, the daughter ion is 1294.65m/z,1157.59m/z and 1100.57m/z, and the corresponding collision energy of the daughter ion is 27V.
The embodiment of the application also provides a method for relatively quantitatively analyzing the 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 of the pig, which applies the peptide fragment composition as described in any one of the preceding items and adopts a liquid chromatography-mass spectrometry combined method to relatively quantitatively analyze the 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 of the liver tissue of the pig.
In some embodiments, the performing a relative quantitative analysis on the 10-formyltetrahydrofolate dehydrogenase ALDH1L1 of the porcine liver tissue by using a liquid chromatography-mass spectrometry combined method comprises:
providing different groups of pig liver samples to be detected, and respectively carrying out protein extraction and proteolysis treatment to obtain different groups of peptide fragments to be detected;
respectively detecting the peptide fragment compositions of different groups by a liquid chromatography-mass spectrometry combined method;
and comparing the detection results of the peptide fragment compositions of different groups to relatively quantify the 10-formyltetrahydrofolate dehydrogenase ALDH1L1 in the pig tissue samples to be detected of different groups.
In some embodiments, the detecting the different groups of peptide fragment compositions by the liquid chromatography-mass spectrometry combined method specifically includes:
respectively incorporating isotopically labeled internal standard peptide fragments into the peptide fragment compositions of different groups; the weight of the internal standard peptide fragment is the same as the weight of the peptide fragment composition;
and carrying out liquid chromatography-mass spectrometry detection on the obtained peptide fragment composition.
In some of these embodiments, the liquid chromatography-mass spectrometry detection is high performance liquid chromatography-tandem mass spectrometry.
In some of these embodiments, each of the different groups contains 4 biological replicate samples.
The embodiments of the present application also provide a kit for relatively quantitative analysis of porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1, said kit comprising reagents for detecting the peptide fragment composition according to any one of claims 1 to 2.
In some embodiments, the reagent comprises a standard of the first peptide fragment, a standard of the second peptide fragment, a standard of the third peptide fragment, a dithiothreitol solution, an iodoacetamide solution, and a trypsin solution.
From the above, the peptide fragment composition, the method and the kit for relatively quantitatively analyzing the porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1 provided by the application can specifically detect and quantitatively analyze the ALDH1L1 by using a liquid chromatograph-mass spectrometer at the protein level through the peptide fragment composition comprising the first peptide fragment, the second peptide fragment and the third peptide fragment, can quickly and efficiently quantitatively detect the porcine ALDH1L1, and improve the detection flux and efficiency of the porcine ALDH1L1. Compared with an antibody-based Western Blot method, the method has stronger specificity, avoids the complex steps, the long period and the high cost for preparing the ALDH1L1 monoclonal antibody, and also avoids the problems of more serious cross reaction, low success rate and the like for preparing the ALDH1L1 polyclonal antibody.
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In order to more clearly illustrate the technical solutions in the present application or related technologies, the drawings required for the embodiments or related technologies in the following description are briefly introduced, and it is obvious that the drawings in the following description are only the embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a flow chart of a method for relatively quantitatively analyzing porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1 according to the present embodiment;
FIG. 2 is a secondary mass spectrum of the first peptide stretch ANATEFGLASGVFTR of example 2;
FIG. 3 is a secondary mass spectrum of second peptide segment DLGEAALNEYLR of example 2;
FIG. 4 is a second mass spectrum of the third peptide segment DTNHGPQNHQAHLR of example 2;
FIG. 5 is a boxplot of the relative quantification results of the ALDH1L1 protein of each treatment group and control group in example 2;
fig. 6 is a boxplot of the relative quantification results of the ALDH1L1 protein for each treatment group and control group in comparative example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail below with reference to the accompanying drawings in combination with specific embodiments.
It should be noted that technical terms or scientific terms used in the embodiments of the present application should have a general meaning as understood by those having ordinary skill in the art to which the present application belongs, unless otherwise defined. The use of "first," "second," and similar terms in the embodiments of the present application do not denote any order, quantity, or importance, but rather the terms are used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that the element or item listed before the word covers the element or item listed after the word and its equivalents, but does not exclude other elements or items.
10-formyl-tetrahydrofolate dehydrogenase ALDH1L1 is a tetramer composed of identical subunits, each subunit composed of three functional domains derived from unrelated genes. Increased ALDH1L1 expression often leads to increased NADPH levels and increased NADPH oxidase activity, resulting in increased ROS levels. In addition, ALDH1L1 appears to be a major regulator of cellular metabolism, and the concentration of ALDH1L1 is strongly down-regulated under certain physiological and pathological conditions, in which case up-regulation of the concentration of ALDH1L1 produces a strong anti-proliferative effect. Therefore, the 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 is a potential marker of a candidate tumor suppressor and invasive cancer, and has a regulation function and higher research value in the oxidative stress related physiological and pathological processes.
In the pig raising industry, the expression increase of ALDH1L1 is correlated with the increase of ROS level, and after the ROS generated by oxidative stress in a pig body is excessively accumulated, the ROS attacks biomacromolecules such as lipid, protein, nucleic acid and the like, so that the components of cells are damaged, the structure of the cells is changed, and the functions are correspondingly changed. On the other hand, the chain reaction of lipid peroxidation can cause the damage of the structure and the related functions of the biological membrane, thereby intensifying the oxidative damage and the apoptosis in the pig body. As two major sites of folate metabolism, ALDH1L1 levels are particularly high in the liver and kidneys of pigs. Therefore, the increased expression of ALDH1L1 is closely related to oxidative stress caused by ROS accumulation, and the oxidative stress further causes inflammatory reaction in pigs and causes oxidation of lipid in livers of the pigs, so that the diseases such as fatty infiltration, liver enlargement, fatty liver, hepatic fibrosis and the like are caused, and the health of the pigs is damaged.
Some assays for determining the relative amount of ALDH1L1 in vivo mainly involve assays at the gene and protein levels. The gene level is mainly analyzed on the mRNA level, for example, the RNA in a tissue is extracted and then is subjected to reverse transcription to form cDNA, a cDNA template after the reverse transcription is amplified according to a specific primer pair designed by the gene of the ALDH1L1, and the transcription level of the ALDH1L1 in the template is compared and analyzed by using a fluorescent real-time quantitative PCR technology. However, for the comparison at the mRNA level, after the gene transcription, the gene is often subjected to regulation such as splicing and splicing to be expressed into the ALDH1L1 protein, so the analysis of the expression level at the transcription level cannot truly reflect the expression level of the gene product protein. On the other hand, methods such as enzyme-linked immunosorbent assay, western Blot, and in situ tissue hybridization are generally used for analyzing ALDH1L1 at the protein level, but these detection methods require high-quality antibodies to specifically recognize ALDH1L1. However, most of the antigen proteins for preparing antibodies in the current market are derived from model organisms such as human beings, mice and rats, and the expression level of the porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1 cannot be quantitatively analyzed on the protein level.
The method for quantitatively analyzing the porcine ALDH1L1 at the protein level can be established by preparing antibodies with strong specificity, such as monoclonal antibodies, polyclonal antibodies and the like, for recognizing the porcine ALDH1L1. However, monoclonal antibodies have problems of long preparation period, high cost and the like; polyclonal antibodies, although less expensive, have a more severe cross-reactivity and a lower success rate.
Therefore, the existing analysis of the swine ALDH1L1 has the problems of inaccurate detection, complex antibody preparation, high cost and the like.
Based on this, the embodiment of the application provides the peptide fragment composition for relatively quantitatively analyzing the porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1 and the application thereof, the peptide fragment composition is used for relatively quantitatively analyzing the porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1 on the protein level, an antibody does not need to be prepared, the specificity is good, and the problems that the detection is not accurate enough, the antibody preparation complexity and the cost are high and the like in the existing analysis of the porcine-derived ALDH1L1 can be solved to a certain extent.
Table 1 shows the relative quantification of the peptide fragment composition of porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1 provided in the examples of the present application.
Referring to table 1, the present application provides a peptide composition for relatively quantitatively analyzing porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1, including a first peptide, a second peptide and a third peptide. Wherein the amino acid sequence of the first peptide segment is shown as SEQ ID NO. 1; the amino acid sequence of the second peptide segment is shown as SEQ ID NO. 2; the amino acid sequence of the third peptide segment is shown as SEQ ID NO. 3.
TABLE 1 peptide fragment composition for the relative quantitative analysis of porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1
Figure BDA0003663122120000051
The first peptide fragment, the second peptide fragment and the third peptide fragment in the peptide fragment composition for relatively quantitatively analyzing the pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 provided by the embodiment of the application are specifically detected by using a liquid chromatograph-mass spectrometer, and the ALDH1L1 can be quantitatively analyzed on the protein level, so that the pig source ALDH1L1 can be rapidly and efficiently relatively quantitatively detected, and the detection flux and efficiency of the pig source ALDH1L1 can be improved. Compared with the detection of the porcine ALDH1L1 by the Western Blot method based on the antibody, the detection method has stronger specificity, can avoid the complex steps, the long period and the high cost for preparing the porcine ALDH1L1 monoclonal antibody, and also avoids the problems of serious cross reaction, low success rate and the like for preparing the porcine ALDH1L1 polyclonal antibody.
Table 2 shows the mass-to-charge ratio information provided in the examples of the present application for the relative quantitative analysis of the peptide fragment composition of porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1.
TABLE 2 Mass/Charge ratio information for relatively quantitative analysis of peptide fragment composition of porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1
Figure BDA0003663122120000061
Referring to table 2, in the peptide fragment composition for quantitative analysis of porcine 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 provided in the embodiments of the present application, the parent ion of the first peptide fragment is 770.89m/z, the daughter ions are 1054.57m/z,907.50m/z and 737.39m/z, and the corresponding collision energy of the daughter ions is 27V. The parent ions of the second peptide fragment are 682.35m/z, the daughter ions are 1135.57m/z,949.51m/z and 878.47m/z, and the corresponding collision energy of the daughter ions is 27V; the parent ion of the third peptide segment is 812.89m/z, the daughter ion is 1294.65m/z,1157.59m/z and 1100.57m/z, and the corresponding collision energy of the daughter ion is 27V. The mass-to-charge ratio, the collision energy and the like of parent ions and daughter ions of the peptide fragment composition can be used for peptide fragment separation and signal acquisition in a High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis method.
Based on the same inventive concept, the present embodiments also provide a kit for relatively quantitative analysis of porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1, comprising reagents for detecting the peptide fragment composition as described in any one of the preceding claims.
In some embodiments, the reagents may include a first peptide fragment standard, a second peptide fragment standard, a third peptide fragment standard, a protein extraction reagent, and a proteolytic reagent.
The protein extraction reagent can be a common protein lysate in the field, such as an SDT protein lysate. The proteolytic reagent can be a conventional proteolytic reagent in the field, such as a dithiothreitol solution, an iodoacetamide solution, a trypsin solution and the like.
The kit of the above embodiment is used for detecting the peptide fragment composition of the corresponding relatively quantitative analysis pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 in any of the foregoing embodiments, and has the beneficial effects of the corresponding peptide fragment composition embodiment, which are not described herein again.
Based on the same inventive concept, the embodiments of the present application further provide a method for relatively quantitatively analyzing the porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1, wherein the peptide fragment composition described in any of the previous embodiments is applied to relatively quantitatively analyze the porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1.
FIG. 1 shows a flow chart of a method for relatively quantitatively analyzing porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1 according to an embodiment of the present application.
As shown in fig. 1, the method for relatively quantitatively analyzing porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1 provided in the embodiments of the present application may include:
s100, providing different groups of pig liver samples to be detected, and respectively carrying out protein extraction and proteolysis treatment to obtain different groups of peptide fragments to be detected;
s200, respectively detecting the peptide fragment compositions of different groups by a liquid chromatography-mass spectrometry combined method;
s300, comparing the detection results of the peptide fragment compositions of different groups to perform relative quantification on the 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 in the pig tissue samples to be detected of different groups.
In some embodiments, in step S100, the protein extraction may include: and extracting the protein in the pig liver sample to be detected by adopting SDT lysate to obtain a protein extract, and quantifying the protein.
In some embodiments, the proteolytic treatment may comprise: and (3) treating the obtained protein extract with dithiothreitol to open a disulfide bond, treating acetamide to block free sulfydryl in the protein, and performing enzyme digestion treatment with trypsin to obtain a peptide fragment.
That is, the protease treatment includes:
treating the protein extract with dithiothreitol to open disulfide bonds to obtain a first product;
treating the first product with acetamide to block free sulfhydryl groups in the protein to obtain a second product;
and (3) digesting the second product by using trypsin.
In some embodiments, in step S200, the method may include:
screening a peptide fragment composition for relatively quantitatively analyzing the 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 of the pig;
the peptide fragment compositions were identified in a targeted manner and corrected by isotopically labeled internal standards.
In some embodiments, the peptide fragment composition of porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1 can be screened for relative quantitation by a high performance liquid chromatography-mass spectrometry combination. Wherein, the conditions of the high performance liquid chromatography-mass spectrometry combined method are as follows:
chromatographic conditions are as follows: a chromatographic column: a C18 column; mobile phase A:0.1% acetonitrile in water, mobile phase B:0.1% aqueous acetonitrile formate; gradient elution procedure: 0-2min, 5-10% of solution B; 2-45min, 10% -30% of solution B; 45-55min, 30-100% of solution B; 55-60min, 100 percent of solution B; flow rate: 250-450 nl/min;
mass spectrum conditions: collecting data in a positive ion mode; primary mass spectrum scanning range: 300-1800m/z, first-order mass spectrum resolution: 60000 (m/z 200), AGC target:3e6, primary Maximum IT:50ms; secondary mass spectrometry: MS2scans:20; isolation window:1.6Th, secondary mass spectral resolution: 15000 (m/z 200), AGC target:1e5, secondary Maximum IT:50ms, MS2 Activation Type: HCD, collision energy: 27V.
In some embodiments, the screening method for screening the peptide fragment composition of the porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1 is repeated three or more times, so that the method for relatively quantitatively analyzing the porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1 has reliable reproducibility. And specific peptide fragment information for realizing accurate and relative quantification of the protein is obtained by comparing, screening and calculating the peptide fragment chromatographic peak and other information obtained by mass spectrometry.
Through the operation, the peptide fragment sequence of the ALDH1L1 protein in the mixed sample is subjected to targeted monitoring, a data acquisition method can be established and optimized, the specific peptide fragment of the target protein ALDH1L1 can be identified through the established data acquisition mode, and the consistency of signal response among all technical repeats is preliminarily evaluated. And determining that ANATEFGLASGVFTR peptide segment with the amino acid sequence number of SEQ ID NO.1, DLGEAALNEYLR peptide segment with the amino acid sequence number of SEQ ID NO.2 and DTNHGPQNHQAHLR peptide segment with the amino acid sequence number of SEQ ID NO.3 can be obtained (namely, the peptide segment composition for relatively quantitatively analyzing the porcine 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 shown in the table 1 is obtained). And simultaneously determining the mass-to-charge ratio information, collision energy and the like of the son-mother ion pairs capable of obtaining the three specific peptide fragments (namely obtaining the mass-to-charge ratio information of the peptide fragment composition of the pig 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 in relative quantitative analysis shown in the table 2).
In some embodiments, targeted identification of the peptide fragment composition and correction by isotopically labeled internal standards may specifically include:
and (4) respectively taking preset quantities of different groups of peptide fragments to be detected in the step S100, doping the internal standard peptide fragments marked by the heavy isotopes with equal quantity for detection, and separating the peptide fragments by adopting LC/MS/MS and collecting signals. In the step, a target peptide fragment parallel reaction monitoring result can be obtained. The result may include information such as peptide fragment chromatographic peaks, raw peak areas, and a histogram of the raw peak areas.
The conditions of the high performance liquid chromatography-mass spectrometry combined method are that
Chromatographic conditions are as follows: a chromatographic column: a C18 column; mobile phase A:0.1% acetonitrile in water, mobile phase B:0.1% aqueous acetonitrile formate solution acetonitrile solution of formic acid; gradient elution procedure: 0-2min, 5-10% of solution B; 2-45min, 10% -30% of solution B; 45-55min, 30-100% of solution B; 55-60min, 100% of solution B; flow rate: 250-450 nl/min;
mass spectrum conditions: collecting data in a positive ion mode; primary mass spectrum scanning range: 300-1800m/z, mass spectral resolution: 60000 (m/z 200), AGC target:3e6, maximum IT:200ms; MS2scans:20; isolation window:1.6Th, mass spectral resolution: 30000 (m/z 200), AGC target:3e6, maximum IT:120ms, MS2 Activation Type: HCD, collision energy: 27V.
The three primary and secondary ion-to-mass-charge ratio information of the specific polypeptide are respectively set as follows: m/z 770.89 is a parent ion of ANATEFGLASGVFTR, a daughter ion produced by fragmentation m/z 1054.57, m/z907.50, m/z 737.39; m/z 682.35 is the parent ion of DLGEAALNEYLR, the daughter ion produced by fragmentation is m/z 1135.57, m/z 949.51, m/z 878.47; m/z 812.89 is the parent ion of DTNHGPQNHQAHLR, and the daughter ion from fragmentation is m/z 1294.65, m/z 1157.59, m/z 1100.57.
In some embodiments, the isotopically labeled internal standard peptide fragment can be PRTC: SAAGAFGPELSR ( 13 C 6 15 N 4 ,+10Da)。
In step S300, the ALDH1L1 in the pig tissue samples to be tested of different groups can be relatively quantified by analyzing the peptide fragment chromatographic peak obtained by parallel reaction monitoring, the original peak area, and the histogram of the original peak area, and comparing the detection results of the peptide fragment compositions of different groups.
Specifically, data analysis is performed on the original file by using the Skyline software, and 3 sub-ions with high abundance of the peptide segment and continuous as much as possible can be selected for quantitative analysis, and the peak areas of the sub-ions of the target peptide segment (that is, the peak areas of the sub-ions of the first peptide segment ANATEFGLASGVFTR, the second peptide segment DLGEAALNEYLR and the third peptide segment DTNHGPQNHQAHLR obtained in step S200) are integrated to obtain the original peak area of the peptide segment in the sample; then, the peak areas of heavy isotope labeled internal standard peptide fragments (namely, the ion peak areas of the first peptide fragment ANATEFGLASGVFTR, the second peptide fragment DLGEAALNEYLR and the third peptide fragment DTNHGPQNHQAHLR with heavy isotope labeling) are used for correction, and the relative expression quantity information of the first peptide fragment ANATEFGLASGVFTR, the second peptide fragment DLGEAALNEYLR and the third peptide fragment DTNHGPQNHQAHLR in samples of different groups is obtained respectively; and finally, calculating the average value of the relative expression quantity of the target peptide in each group of samples, and performing statistical analysis. And analyzing the expression quantity of the target protein ALDH1L1, and further calculating to obtain the relative expression quantity difference of the target protein in different sample groups according to the relative expression quantity of the corresponding peptide segment of each target protein in different sample groups. The specific calculation is the prior art, and may be implemented by, for example, skyline software, so that the detailed calculation steps and the like are not described herein again.
The method of the above embodiment is used to realize the detection of the peptide fragment composition of the porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1 in any one of the foregoing embodiments, and has the beneficial effects of the corresponding embodiment of the peptide fragment composition, which are not described herein again.
It should be noted that the above describes some embodiments of the present application. Other embodiments are within the scope of the following claims. In some cases, the actions or steps recited in the claims may be performed in a different order than in the embodiments described above and still achieve desirable results. In addition, the processes depicted in the accompanying figures do not necessarily require the particular order shown, or sequential order, to achieve desirable results. In some embodiments, multitasking and parallel processing may also be possible or may be advantageous.
The technical solution of the present invention will be further described with reference to the following embodiments.
The experimental procedures in the following examples are conventional unless otherwise specified.
The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
EXAMPLE 1 screening for the relative quantitative analysis of the peptide fragment composition of porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1
1. Extracting ALDH1L1 protein: pig liver tissue samples of a diquat induced oxidative stress treatment group (DQ), an antioxidant lipoic acid treatment group (LA), a diquat + lipoic acid treatment group (DL) and a control group (CK) are respectively taken, added with a proper amount of SDT lysate, transferred into a 2ml centrifuge tube filled with a proper amount of quartz sand in advance, and homogenized and crushed by using a homogenizer (24 x 2,6.0M/S,60S, twice). Then, the ultrasonic wave (100W, working time 10s, intermittent time 10s, circulation time 10 times) is carried out, and the water is boiled for 10min. Centrifuging at 14000g for 10min, collecting supernatant, filtering with 10kD ultrafiltration membrane, and collecting filtrate. Protein quantification was performed using BCA method. Samples were aliquoted and stored at-80 ℃. Each group contained 4 biological replicates.
2. And (3) proteolysis: in each treatment group and the control group, about 200ug of protein was sampled from each group, dithiothreitol (DTT) was added to a final concentration of 100mM to open disulfide bonds, followed by 15min in a boiling water bath, cooling to room temperature, adding 200. Mu.L of urea buffer (UA buffer,8M Urea,150mM Tris-HCl, pH 8.0), mixing well, transferring to a 10KD ultrafiltration tube, and centrifuging 14000g 30min. Add 200. Mu.l of UA buffer and centrifuge 14000g 30min, discard the filtrate. Add 100. Mu.L iodoacetamide (IAA, 50mM IAA in UA) to alkylate the free thiol groups inside the blocked protein, shake at 600rpm for 1min, protect from light for 30min at room temperature, and centrifuge for 14000g for 20min. Add 100. Mu.L of UA buffer and centrifuge 14000g for 20min for 3 times. Add 100. Mu.L NH 4 HCO 3 buffer (50 mM), centrifuge 14000g 20min for 2 times. Add 40. Mu.L NH 4 HCO 3 buffer (containing Trypsin in a 1-enzyme ratio), shaking at 600rpm for 1min, and 16h at 37 ℃. The collection tube was replaced with a fresh one and centrifuged for 14000g 15min. Add 40. Mu.L NH 4 HCO 3 The buffer (50 mM) was centrifuged at 14000g 30min and the filtrate collected. The peptide fragment after enzymolysis is desalted and lyophilized, then is redissolved by 0.1 percent Formic Acid (FA), and the concentration of the peptide fragment is determined by OD 280.
3. Screening the specific peptide fragment of the target protein and establishing a data acquisition method: firstly, randomly selecting a sample from a plurality of repetitions of each group in the step 2, respectively taking a proper amount of peptide fragments after enzymolysis, and equivalently mixing the peptide fragments into a sample; taking 1ug of mixed peptide fragment, and separating by HPLC; the buffer solution A is 0.1% formic acid aqueous solution, and the solution B is 0.1% formic acid acetonitrile aqueous solution (acetonitrile is 84%); the chromatographic column is balanced by 95 percent of solution A; samples were run through a Trap Column (100 μm. By 50mm,5 μm-C18, dr. Maisch phases, r25. Aq), and an Analytical Column (180 μm. By 150mm,3 μm-C18, dr. Maisch phases, r23. Aq) at a flow rate of 300nl/min; the liquid phase separation gradient was as follows: from 0min to 2min, linear gradient of liquid B from 5% to 10%, from 2min to 45min, linear gradient of liquid B from 10% to 30%; 45-55 minutes, linear gradient of B liquid from 30% to 100%; the linear gradient of the liquid B is maintained at 100 percent within 55-60 minutes. Then, performing targeted qualitative analysis by using a Q-active HF mass spectrometer through a parallel reaction monitoring method; analysis duration: 60min, detection mode: positive ion, parent ion scan range: 300-1800m/z, first order mass spectral resolution: 60000 (m/z 200), AGC target:3e6, primary Maximum IT:50ms; peptide fragment secondary mass spectrometry was collected as follows: acquisition of 20 secondary mass spectra (MS 2 scan) was triggered after each full scan (full scan), secondary mass resolution: 15000 (m/z 200), AGC target:1e5, secondary Maximum IT:50ms, MS2 Activation Type: HCD, isolation window:1.6Th, normalized collagen energy:27; and performing MS2 scanning on the candidate peptide fragment of the target protein by LC-MS/MS by adopting a targeted shotgun scanning mode.
By comparing, screening and calculating the information such as peptide fragment chromatographic peaks obtained by mass spectrometry, determining that the sequence information of the peptide fragment composition used for relatively quantitatively analyzing the pig 10-formyltetrahydrofolate dehydrogenase ALDH1L1 in the table 1 can be obtained in all treatment groups, determining the mass-to-charge ratio information of the peptide fragment composition used for relatively quantitatively analyzing the pig 10-formyltetrahydrofolate dehydrogenase ALDH1L1 in the table 2, and determining the specific steps and the specific parameter conditions of the steps of the method for relatively quantitatively analyzing the pig 10-formyltetrahydrofolate dehydrogenase ALDH1L1.
Example 2 application of liver ALDH1L1 protein relativity quantification of aquacide induced oxidative stress fattening pig model
1. Extracting ALDH1L1 protein: pig liver tissue samples of a diquat induced oxidative stress treatment group (DQ), an antioxidant lipoic acid treatment group (LA), a diquat + lipoic acid treatment group (DL) and a control group (CK) are respectively taken, added with a proper amount of SDT lysate, transferred into a 2ml centrifuge tube filled with a proper amount of quartz sand in advance, and homogenized and crushed by using a homogenizer (24 x 2,6.0M/S,60S, twice). Then, the ultrasonic wave (100W, working time 10s, intermittent time 10s, circulation time 10 times) is carried out, and the water is boiled for 10min.14000g, centrifuging for 10min, taking the supernatant, filtering by a 10kD ultrafiltration membrane, and collecting the filtrate. Protein quantification was performed using BCA method. Samples were aliquoted and stored at-80 ℃. Each group contained 4 biological replicates.
2. And (3) proteolysis: in each treatment group and the control group, about 200ug of protein was sampled from each group, dithiothreitol (DTT) was added to a final concentration of 100mM to open disulfide bonds, followed by 15min in a boiling water bath, cooling to room temperature, adding 200. Mu.L of urea buffer (UA buffer,8M Urea,150mM Tris-HCl, pH 8.0), mixing well, transferring to a 10KD ultrafiltration tube, and centrifuging 14000g 30min. Add 200. Mu.l of UA buffer and centrifuge 14000g 30min, discard the filtrate. Add 100. Mu.L iodoacetamide (IAA, 50mM IAA in UA) to alkylate the free thiol groups inside the blocked protein, shake at 600rpm for 1min, protect from light for 30min at room temperature, and centrifuge for 14000g for 20min. Add 100. Mu.L of UA buffer, centrifuge 14000g for 20min and repeat 3 times. Add 100. Mu.L of NH 4 HCO 3 buffer (50 mM), centrifuge 14000g 20min for 2 times. Add 40. Mu.L NH 4 HCO 3 buffer (containing Trypsin in a 1-enzyme ratio), shaking at 600rpm for 1min, and 16h at 37 ℃. The collection tube was replaced with a fresh one and centrifuged for 14000g 15min. Add 40. Mu.L NH 4 HCO 3 The buffer (50 mM) was centrifuged at 14000g 30min and the filtrate collected. The peptide fragments after enzymolysis are desalted and lyophilized, then are redissolved by 0.1 percent Formic Acid (FA), and OD280 is used for determining the concentration of the peptide fragments.
3. Target identification of target protein specific peptide fragments and correction by isotopic internal standard: firstly, about 1ug of peptide fragment is taken from each sample in a plurality of repetitions of each group in the step 2, 20fmol heavy isotope labeled internal standard peptide fragments (PRTC: SAAGAFGPELSR) are respectively doped for detection, and an HPLC system is adopted to carry out high performance liquid chromatography separation on the polypeptide; the buffer solution A is 0.1% formic acid aqueous solution, and the solution B is 0.1% formic acid acetonitrile aqueous solution (acetonitrile is 84%); the chromatographic column is balanced by 95 percent of solution A; injecting a sample into a chromatographic analysis column for gradient separation, wherein the flow rate is 300nl/min; the liquid phase separation gradient was as follows: from 0min to 2min, linear gradient of liquid B from 5% to 10%, from 2min to 45min, linear gradient of liquid B from 10% to 30%; 45-55 minutes, linear gradient of B liquid from 30% to 100%; the linear gradient of the liquid B is maintained at 100 percent within 55-60 minutes. After the high performance liquid chromatography separation, performing parallel reaction monitoring mass spectrometry on five target peptide segments of the identified target protein by using a Q-active HF mass spectrometer, wherein the analysis time is as follows: 60min, detection mode: a positive ion; primary mass spectrum scanning range: 300-1800m/z, mass spectral resolution: 60000 (m/z 200), AGC target:3e6, maximum IT:200ms; after each one-stage MS scan (full MS scan), 20 parallel reaction monitoring scans (MS 2 scans) were collected according to the Inclusion list, isolation window:1.6Th, mass spectral resolution: 30000 (m/z 200), AGC target:3e6, maximum IT:120ms, MS2 Activation Type: HCD, normalized fusion energy:27.
setting the mass-to-charge ratio information of three primary and secondary ions of the specific polypeptide as follows: m/z 770.89 is a parent ion of ANATEFGLASGVFTR, a daughter ion m/z 1054.57, m/z907.50, m/z 737.39 produced by fragmentation; m/z 682.35 is the parent ion of DLGEAALNEYLR, the daughter ion produced by fragmentation is m/z 1135.57, m/z 949.51, m/z 878.47; m/z 812.89 is the parent ion of DTNHGPQNHQAHLR, and the daughter ions from fragmentation are m/z 1294.65, m/z 1157.59, m/z 1100.57.
4. Analyzing the monitoring result of the parallel reaction of the target peptide fragment: and analyzing the target protein specific peptide segment data obtained by parallel reaction monitoring, wherein the target protein specific peptide segment data comprises information such as a peptide segment chromatographic peak, an original peak area, a comparison histogram of the original peak area and the like. 3 daughter ions with high abundance of peptide fragments and continuous as much as possible are selected for quantitative analysis. Firstly, integrating the peak areas of the daughter ions of a target peptide fragment to obtain the original peak area of the peptide fragment in a sample; then, correcting the peak area of the heavy isotope labeled internal standard peptide segment to obtain the relative expression amount information of each segment of peptide in different samples; and finally, calculating the average value of the relative expression quantity of the target peptide in each group of samples, and performing statistical analysis. And analyzing the expression quantity of the target protein, and further calculating to obtain the relative expression quantity difference of the target protein in different sample groups according to the relative expression quantity of the corresponding peptide segment of each target protein among different sample groups.
And (3) test results: as shown in fig. 2-5. In the diquat + lipoic acid treatment group (DL), the secondary mass spectrum of the first peptide segment ANATEFGLASGVFTR, the secondary mass spectrum of the second peptide segment DLGEAALNEYLR and the secondary mass spectrum of the third peptide segment DTNHGPQNHQAHLR are shown in fig. 2, fig. 3 and fig. 4 respectively.
Fig. 5 is a relative quantification result of ALDH1L1 protein in the control group (CK) compared to the average value of ALDH1L1 protein in the three treatment groups (diquat induced oxidative stress treatment group (DQ), antioxidant lipoic acid treatment group (LA), and diquat + lipoic acid treatment group (DL)). DL is the relative quantification of ALDH1L1 protein in diquat + lipoic acid treated group (DL) relative to ALDH1L1 protein in control group (CK). DQ is the relative quantification of ALDH1L1 protein in the diquat induced oxidative stress treated group (DQ) versus ALDH1L1 protein in the control group (CK). LA is the relative quantification of ALDH1L1 protein in the antioxidant lipoic acid treated group (LA) relative to ALDH1L1 protein in the control group (CK).
Comparative example 1
The difference from the embodiment 2 is that based on the LC-MS/MS method, the peptide fragment after enzymolysis is detected by adopting the TMT (Tandem Mass Tag) protein isotope labeling quantitative technology according to the specific operation instruction of the TMT protein labeling kit of Thermo company.
Specifically, the peptide fragments after enzymolysis are obtained through step 1 and step 2 in example 1.
And (3) test results: see fig. 6.
CK in fig. 6 is a relative quantitative result of ALDH1L1 in the control group (CK) compared to the average value of ALDH1L1 in the three treatment groups (diquat induced oxidative stress treatment group (DQ), antioxidant lipoic acid treatment group (LA), and diquat + lipoic acid treatment group (DL)). DL is the relative quantification of ALDH1L1 in diquat + lipoic acid treated group (DL) relative to ALDH1L1 in control group (CK). DQ is the relative quantification of ALDH1L1 in the diquat induced oxidative stress treated group (DQ) relative to ALDH1L1 in the control group (CK). LA is the relative quantification of ALDH1L1 in the antioxidant lipoic acid treated group (LA) relative to ALDH1L1 in the control group (CK).
Comparing the relative quantitative results boxplot of fig. 5 obtained in example 2 with that of fig. 6 obtained in comparative example 1, it can be seen that the trend of the ALDH1L1 identified in example 2 among the different treatment groups is consistent with the trend of the ALDH1L1 identified in comparative example 1 among the different treatment groups. It is shown that the relative quantitative analysis pig including the peptide fragment composition for the relative quantitative analysis of the pig-derived 10-formyltetrahydrofolate dehydrogenase A has good accuracy, and the peptide fragment composition can be used for identifying the pig 10-formyltetrahydrofolate dehydrogenase ALDH1L1 in a targeted manner, and has higher sensitivity compared with the high-throughput identification of all proteins of the pig by the existing in vitro labeled detection method, such as the TMT (Tandem Mass Tag) protein isotope labeling quantitative technology.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the present disclosure, also technical features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in detail for the sake of brevity.
While the present disclosure has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations thereof will be apparent to those skilled in the art in light of the foregoing description.
The disclosed embodiments are intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalents, improvements, and the like that may be made within the spirit and principles of the embodiments of the disclosure are intended to be included within the scope of the disclosure.
Sequence listing
<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
<120> peptide fragment composition for relatively quantitative analysis of porcine 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 and application thereof
<130> FI221035
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213> amino acid sequence of first peptide fragment
<400> 1
Ala Asn Ala Thr Glu Phe Gly Leu Ala Ser Gly Val Phe Thr Arg
1 5 10 15
<210> 2
<211> 12
<212> PRT
<213> amino acid sequence of second peptide fragment
<400> 2
Asp Leu Gly Glu Ala Ala Leu Asn Glu Tyr Leu Arg
1 5 10
<210> 3
<211> 14
<212> PRT
<213> amino acid sequence of third peptide fragment
<400> 3
Asp Thr Asn His Gly Pro Gln Asn His Gln Ala His Leu Arg
1 5 10

Claims (9)

1. The peptide fragment composition for relatively quantitatively analyzing the porcine 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 is characterized by comprising a first peptide fragment, a second peptide fragment and a third peptide fragment;
wherein the amino acid sequence of the first peptide segment is shown as SEQ ID NO. 1; the amino acid sequence of the second peptide segment is shown as SEQ ID NO. 2; the amino acid sequence of the third peptide segment is shown as SEQ ID NO. 3.
2. The peptide fragment composition for relatively quantitatively analyzing the porcine 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 according to claim 1, wherein the parent ion of the first peptide fragment is 770.89m/z, the daughter ions are 1054.57m/z,907.50m/z and 737.39m/z, and the corresponding collision energy of the daughter ions is 27V; the parent ions of the second peptide segment are 682.35m/z, the daughter ions are 1135.57m/z,949.51m/z and 878.47m/z, and the corresponding collision energy of the daughter ions is 27V; the parent ion of the third peptide segment is 812.89m/z, the daughter ion is 1294.65m/z,1157.59m/z and 1100.57m/z, and the corresponding collision energy of the daughter ion is 27V.
3.A method for relatively quantitatively analyzing 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 of pigs, which is characterized in that the peptide fragment composition of any one of claims 1 to 2 is used for relatively quantitatively analyzing 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 of pig liver tissues by a liquid chromatography-mass spectrometry combined method.
4. The method of claim 3, wherein the performing the relative quantitative analysis of the 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 of the porcine liver tissue by the liquid chromatography-mass spectrometry comprises:
providing different groups of pig liver samples to be detected, and respectively carrying out protein extraction and proteolysis treatment to obtain different groups of peptide fragments to be detected;
respectively detecting the peptide fragment compositions of different groups by a liquid chromatography-mass spectrometry combined method;
comparing the detection results of the peptide fragment compositions of different groups to perform relative quantification on the 10-formyl tetrahydrofolate dehydrogenase ALDH1L1 in the pig tissue samples to be detected of different groups.
5. The method according to claim 4, wherein the detecting the different groups of peptide fragment compositions by the liquid chromatography-mass spectrometry combination method comprises:
respectively incorporating isotopically labeled internal standard peptide fragments into the peptide fragment compositions of different groups; the weight of the internal standard peptide fragment is the same as the weight of the peptide fragment composition;
and carrying out liquid chromatography-mass spectrometry detection on the obtained peptide fragment composition.
6. The method of claim 5, wherein the liquid chromatography-mass spectrometry detection is high performance liquid chromatography-tandem mass spectrometry.
7. The method of claim 4, wherein each of the different groups contains 4 biological replicates.
8. A kit for relatively quantitatively analyzing porcine 10-formyltetrahydrofolate dehydrogenase ALDH1L1, comprising reagents for detecting the peptide fragment composition of any one of claims 1-2.
9. The kit of claim 8, wherein the reagents comprise a standard for the first peptide fragment, a standard for the second peptide fragment, a standard for the third peptide fragment, a dithiothreitol solution, an iodoacetamide solution, and a trypsin solution.
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WO2000074711A2 (en) * 1999-06-03 2000-12-14 Pharmaproducts Uk Limited 10-formyltetrahydrofolate dehydrogenase as therapeutical agent
WO2015160470A2 (en) * 2014-03-20 2015-10-22 The Trustees Of Princeton University Nadph production by the 10-formyl-thf pathway, and its use in the diagnosis and treatment of disease

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